by Liang Yiu-hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 104-117). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.iii / AMINO ACIDS SHORTHAND --- p.v / TABLE OF CONTENTS --- p.vi-x / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- The Discovery of IGFs --- p.1 / Chapter 1.3 --- The Growth Promoting Actions of IGFs --- p.3 / Chapter 1.4 --- Molecular Biology of IGFs in Mammals --- p.6 / Chapter 1.4.1 --- IGF Genes and Transcripts --- p.6 / Chapter 1.4.2 --- Regulation of IGF Gene Expression --- p.8 / Chapter 1.5 --- IGF Binding Proteins --- p.11 / Chapter 1.5.1 --- Regulation of IGF Action by IGF Binding Proteins --- p.11 / Chapter 1.6 --- The Insulin and IGF Receptors --- p.13 / Chapter 1.6.1 --- IGF-I Receptor --- p.13 / Chapter 1.6.2 --- IGF-II Receptor --- p.13 / Chapter 1.6.3 --- Insulin/IGF-I Hybrid Receptor --- p.15 / Chapter 1.7 --- IGF in Mammalian Fetal Growth --- p.17 / Chapter 1.8 --- The Role of IGFs in Fish --- p.19 / Chapter 1.9 --- Aims of the Present Study --- p.26 / Chapter CHAPTER 2 --- GENERAL METHODOLOGY / Chapter 2.1 --- Materials --- p.27 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Gene Clean --- p.32 / Chapter 2.2.1A --- Gene Clean by Glassmilk Method --- p.32 / Chapter 2.2.1B --- Gene Clean by Sephaglas´ёØ BandPrep Kit --- p.32 / Chapter 2.2.2 --- Preparation of Radioactive Nucleic Acid Probes --- p.33 / Chapter 2.2.3 --- Sephadex G-50 Spun-column Chromatography --- p.33 / Chapter 2.2.4 --- Small Scale Alkali Preparation of Plasmid DNA --- p.34 / Chapter 2.2.5 --- Large Scale Preparation of Plasmid DNA 36 - using Wizard Maxiprep Kit (Promega) / Chapter 2.2.6 --- DNA Sequencing using T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.37 / Chapter 2.2.7 --- Restriction Enzyme Digestion --- p.38 / Chapter 2.2.8 --- Agarose Gel Electrophoresis --- p.39 / Chapter 2.2.9 --- Dephosphorylation of Linearized Plasmid DNA --- p.39 / Chapter 2.2.10 --- Ligation of Foreign DNA with Linearized Plasmid --- p.40 / Chapter 2.2.11 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.40 / Chapter 2.2.12 --- Blotting : Transfer of DNA to Nylon Membrane --- p.41 / Chapter 2.2.12A --- Capillary Transfer of DNA to Nylon Membrane in 10X SSC --- p.41 / Chapter 2.2.12B --- Capillary Transfer of DNA to Nylon Membrane under Alkaline Condition --- p.42 / Chapter CHAPTER 3 --- SCREENING OF COMMON CARP LIVER cDNA LIBRARY / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Methods --- p.48 / Chapter 3.2.2.1 --- Preparation of the Plating Host --- p.48 / Chapter 3.2.2.2 --- Phage Titering --- p.48 / Chapter 3.2.2.3 --- Primary Screening of Common Carp Liver cDNA Library --- p.48 / Chapter 3.2.2.4 --- Purification of the Positive Clone --- p.49 / Chapter 3.2.2.5 --- Checking the Insert Size of the Positive Clone --- p.50 / Chapter 2.2.2.6 --- In vivo Excision to Release Phagemid from the Phage vector --- p.51 / Chapter 3.2.2.7 --- Plasmid Minipreparation of the Positive Clone --- p.52 / Chapter 3.2.2.8 --- Restriction Enzyme Digestion to Release the Insert --- p.52 / Chapter 3.2.2.9 --- Large Scale Plasmid Preparation of the Positive Clone --- p.53 / Chapter 3.2.2.10 --- DNA Sequencing of the Positive Clone --- p.53 / Chapter 3.2.2.11 --- Restriction Mapping of the Positive Clone --- p.53 / Chapter 3.2.2.12 --- Subcloning of the Positive Clone into Plasmid Vectors --- p.53 / Chapter 3.2.2.13 --- DNA Sequencing of the Subclones --- p.54 / Chapter 3.3 --- Results and Discussion --- p.55 / Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.2 --- Materials and Methods --- p.71 / Chapter 4.2.1 --- Materials --- p.71 / Chapter 4.2.2 --- Methods --- p.72 / Chapter 4.2.2.1 --- Tissue Preparation --- p.72 / Chapter 4.2.2.2 --- Total RNA Extraction --- p.72 / Chapter 4.2.2.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.73 / Chapter 4.2.2.4 --- First Strand cDNA Synthesis --- p.74 / Chapter 4.2.2.5 --- IGF-I Specific PCR --- p.75 / Chapter 4.2.2.6 --- Preparation of Carp IGF Conserve Region --- p.75 / Chapter 4.2.2.7 --- Southern Hybridization of PCR Products --- p.76 / Chapter 4.3 --- Results and Discussion --- p.76 / Chapter CHAPTER 5 --- GENOMIC SOUTHERN ANALYSIS / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and Methods --- p.81 / Chapter 5.2.1 --- Materials --- p.81 / Chapter 5.2.2 --- Methods --- p.82 / Chapter 5.2.2.1 --- Preparation of Genomic DNA from Carp Testis --- p.82 / Chapter 5.2.2.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.82 / Chapter 5.2.2.3 --- Southern Blotting of the Digested Genomic DNA --- p.83 / Chapter 5.2.2.4 --- Preparation of the Carp IGF-I Specific Probe --- p.83 / Chapter 5.2.2.5 --- Genomic Southern Hybridization --- p.83 / Chapter 5.3 --- Results and Discussion --- p.84 / Chapter CHAPTER 6 --- THE SEARCH OF OTHER IGF cDNA SUBTYPES IN COMMON CARP / Chapter 6.1 --- Introduction --- p.88 / Chapter 6.2 --- Materials and Methods --- p.89 / Chapter 6.2.1 --- Materials --- p.89 / Chapter 6.2.2 --- Methods --- p.91 / Chapter 6.2.2.1 --- Screening using a Conserve Region cDNA Probe of Carp IGF-I --- p.91 / Chapter 6.2.2.2 --- PCR using IGF-I Specific Primers --- p.92 / Chapter 6.2.2.3 --- PCR Using T3 and T7 Primers --- p.92 / Chapter 6.2.2.4 --- Southern Blot Analysis of T3 and T7 PCR Products of cDNA Insert --- p.93 / Chapter 6.2.2.5 --- DNA Sequencing of Positive Clones --- p.94 / Chapter 6.3 --- Results and Discussion --- p.94 / Chapter CHAPTER 7 --- GENERAL DISCUSSION AND CONCLUSION --- p.99 / REFERENCES --- p.104-117

by Agnes Pui-Yee Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 162-177). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / ABBREVIATIONS --- p.vii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Physiology of growth --- p.3 / Chapter 1.2 --- The anterior pituitary --- p.4 / Chapter 1.3 --- Chemistry of GH and the GH gene family --- p.7 / Chapter 1.4 --- Biochemical effects and mode of action of GH --- p.8 / Chapter 1.5 --- Control of GH at cellular level --- p.10 / Chapter 1.6 --- Control of GH gene expression at molecular level / Chapter 1.6.1 --- Introduction --- p.11 / Chapter 1.6.2 --- Tissue-specific expression of GH gene / Chapter 1.6.2.1 --- Tissue-specific transcription factors of pituitary cells --- p.20 / Chapter 1.6.2.2 --- Non-tissue specific transcription factors of pituitary cells --- p.27 / Chapter 1.6.2.3 --- Negatively-acting transcription factors of non-pituitary cells --- p.34 / Chapter 1.6.2.4 --- Theory for tissue-specific GH gene activation --- p.39 / Chapter 1.7 --- Characteristic of growth in fish --- p.40 / Chapter 1.8 --- Objectives of the present study --- p.42 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- General techniques / Chapter 2.1.1 --- Preparation of DNA / Chapter 2.1.1.1 --- Minipreparation of DNA --- p.46 / Chapter 2.1.1.2 --- Preparation of DNA using Qiagen column --- p.47 / Chapter 2.1.1.3 --- Preparation of phage DNA --- p.48 / Chapter 2.1.2 --- Elution of DNA from agarose gel --- p.51 / Chapter 2.1.3 --- Preparation of competence cells and transformation --- p.52 / Chapter 2.1.4 --- Ligation of DNA fragments --- p.53 / Chapter 2.1.5 --- Cell feeding and subculturing --- p.54 / Chapter 2.2 --- Special techniques / Chapter 2.2.1 --- DNA sequencing --- p.56 / Chapter 2.2.2 --- Polymerase chain reaction (PCR) --- p.67 / Chapter 2.2.3 --- Direct sequencing of PCR products --- p.72 / Chapter 2.2.4 --- Nested-deletion --- p.75 / Chapter 2.2.5 --- DNA transfection --- p.81 / Chapter 2.2.6 --- CAT assay --- p.86 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Sequencing of the grass carp GH gene / Chapter 3.1.1 --- Introduction --- p.93 / Chapter 3.1.2 --- Sequencing strategy --- p.94 / Chapter 3.2 --- Sequence analysis of the grass carp GH gene --- p.108 / Chapter 3.3 --- Functional analysis of the grass carp GH gene --- p.115 / Chapter CHAPTER 4 --- DISCUSSIONS / Chapter 4.1 --- DNA sequence comparison between grass carp GH gene and other organisms --- p.137 / Chapter 4.2 --- Amino acid comparisons between grass carp GH and other organisms --- p.143 / Chapter 4.3 --- Tissue-specific expression of GH gene / Activation of transcription --- p.154 / Repression of transcription --- p.155 / Chapter 4.4 --- Electroporation of zebrafish eggs --- p.157 / Chapter 4.5 --- Further studies --- p.160 / REFERENCES --- p.162 / APPENDIX --- p.178

by Vong Puinga Queenie Maria. / "August 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 176-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

No description available.

Due to the importance of insulin-like growth factor-I (IGF-I) in regulating many physiological and metabolic processes, the IGF-I gene was chosen as a candidate gene to study trait associated polymorphisms in chickens. A PstI restriction fragment length polymorphism (RFLP) was detected at the 5' region of the gene and mapped to about 7 Kb upstream of the published promoter sequence. Analysis for association of the marker with traits of economic importance in an unselected, random-bred population of 359 White Leghorns revealed a significant association with egg weight (P ≤ 0.05) and specific gravity (P ≤ 0.05). There was also a trend for association with juvenile body weight (P = 0.08) but not adult body weight. For egg weight the PstI (-/-) genotype was associated with lower egg weight as compared to the heterozygote or the PstI (+/+) genotype. The PstI marker also was found to be significantly associated with differences in trait correlations. A regulatory loop that co-ordinated feed consumption, body weight, egg weight and rate of egg laying was detected, and this regulatory loop differed among the IGF-I genotypic classes. In the PstI (+/-) genotype, the degree of correlation between some of the traits was time dependent, while in the PstI (+/+) genotype it remained constant through the different periods of measurement. Since IGF-I is known to play an important role in immune functions, the association of the IGF-I genotypes with immune traits was also investigated. A significant association was found for delayed type hypersensitivity, interferon production and T-cell count (P ≤ 0.05). Individuals belonging to the PstI (+/-) genotypic class exhibited higher immune response, reflected by the delayed type hypersensitivity reaction and antibody the interactive effects of marker genotypes in the GH, GH-receptor and IGF-I genes on traits and trait correlations indicated that the three are part of an epistatic pathway, wherein the phenotypic consequences of

No description available.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

The type 1 insulin-like growth factor receptor (lGF-1 R) plays a significant role in tumor growth and spread, and IGF-1 R inhibitors and antibodies are now undergoing clinical testing. However, factors that regulate sensitivity to IGF-1 R inhibition remain unclear. The aim of this project is to identify proteins whose depletion regulates sensitivity to IGF-1 R inhibition, in order to design effective combination treatments to benefit patients. An IGF-1 R kinase inhibitor, AZ12253801 (provided by AstraZeneca) was able to block IGF-induced phosphorylation of IGF-1 R in DU145 prostate cancer and MCF-7 breast cancer cells, inhibited downstream signalling in DU145 cells, and also inhibited proliferation and cell survival of both cell lines. AZ12253801 was used in an unbiased siRNA screen in both cell lines, using two s'iRNA libraries (779 kinase-related Kinome and 230 DNA repair-associated siRNAs). Eight Kinome and five DNA repair-associated hits have been identified after primary and second round screens, and further validated. The strongest hit was dishevelled homolog 3 (DVL3), a member of the WNT signalling pathway, which is highly expressed in both cell lines. DVL3 silencing caused reduction in active l3-catenin and inactivated the mTOR pathway, consistent with previous studies, and did not affect IGF-1 Rand AKT activity. However, DVL3 silencing led to activation of MEK1/2-ERK1/2 in serum-starved cells and sensitized this pathway to IGF-1 stimulation, with translocation of ERK1/2 into the nucleus and increased expression of ERK1/2 target genes. A DVL PDZ domain inhibitor (DVLi) showed similar effects on active l3-catenin, mTOR signalling and ERK1/2 signalling activity. The administration of DVLi increased sensitivity to AZ12253801 in cell lines with detectable ERK1/2 activation, but not prostate cancer cells in which ERK signalling was suppressed and AKT was activated in the context of loss of functional PTEN. Furthermore, DVL3 regulated activation of ERKs by influencing signaling downstream of the IGF-1 R and upstream of RAS, and DVL3 was found in a complex with the adaptor proteins GRB2 and DAB2. GRB2 knockdown was capable of abolishing ERK1/2 activation induced by DVLi, further implicating involvement of GRB2, and DAB2 silencing sensitized to IGF-1 R inhibition, mimicking effects of DVL3 depletion. Taken together, DVL3 silencing or inhibition enhances sensitivity to IGF-1 R inhibition by negatively regulating the ERK1/2 signaling pathway. These investigations shed new light on the factors that regulate IGF signaling, and provide a rational basis for design of clinical trials of IGF-1 R inhibitors.

by Pong Tsang Wai Hai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 98-105). / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Biological functions and structure of GH --- p.1 / Chapter 1.2 --- Application of recombinant GH --- p.2 / Chapter 1.3 --- Expression of eukaryotic gene in E.coli --- p.4 / Chapter 1.4 --- Methods for increasing expression of a cloned gene --- p.6 / Chapter 1.4.1 --- Changing the 5' end codons of the cDNA to E.coli preferred codons --- p.6 / Chapter 1.4.2 --- Optimization of distance between SD sequence and the initiation codons --- p.6 / Chapter 1.4.3 --- "Construction of a short ""dummy"" cistron at the 5' end of the cloned gene to improve attachment of ribosome" --- p.7 / Chapter 1.4.4 --- Increasing the copy number of recombinant expression plasmid --- p.8 / Chapter 1.4.5 --- Optimizing high density cell expression --- p.9 / Chapter 1.5 --- Quantitating the expression of cloned gene --- p.10 / Chapter 1.6 --- Inclusion bodies formation --- p.11 / Chapter 1.7 --- The purification of eukaryotic polypeptides synthesized as inclusion bodies --- p.12 / Chapter 1.7.1 --- Solubilization of the inclusion bodies --- p.13 / Chapter 1.7.2 --- Refolding the polypetides and disulfide bond formation --- p.13 / Chapter 1.8 --- Expression of secreted recombinant protein --- p.14 / Chapter 1.9 --- Purpose of present study --- p.15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General techniques --- p.16 / Chapter 2.1.1 --- Chemical Synthesis of DNA linkers and primers --- p.16 / Chapter 2.1.2 --- Manipulation of DNA --- p.16 / Chapter 2.1.3 --- Electro-elution of DNA from Agarose Gel --- p.17 / Chapter 2.1.4 --- Preparation of Competent Cells and Transformation --- p.18 / Chapter 2.1.5 --- Screening of the Expressed Clones --- p.19 / Chapter 2.1.6 --- Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.21 / Chapter 2.1.7 --- Western blot analysis --- p.21 / Chapter 2.2 --- Purification procedures --- p.23 / Chapter 2.2.1 --- Growing up the cells in large scale --- p.23 / Chapter 2.2.2 --- Harvesting of cells from large scale culture --- p.23 / Chapter 2.2.3 --- Sonication of the cells --- p.24 / Chapter 2.2.4 --- Washing of the inclusion body --- p.24 / Chapter 2.2.5 --- Solubilization of the inclusion bodies --- p.25 / Chapter 2.2.6 --- Renaturation of r-gcGH --- p.26 / Chapter 2.2.6.1 --- Step down dilution mehtod --- p.26 / Chapter 2.2.6.2 --- Rapid dilution method --- p.26 / Chapter 2.2.7 --- Separation by reverse phase chromatography --- p.27 / Chapter 2.2.7.1 --- Octadodecylsilica (ODS) column separation --- p.27 / Chapter 2.2.7.2 --- Fast performance Liquid Chromatography(FPLC) --- p.28 / Chapter 2.3 --- Characterization methods --- p.29 / Chapter 2.3.1 --- Radioimmunoassay --- p.29 / Chapter 2.3.1.1 --- Iodination of r-gcGH --- p.29 / Chapter 2.3.1.2 --- Binding assay --- p.31 / Chapter 2.3.2 --- Preparation of anti-r-gcGH serum --- p.32 / Chapter 2.3.3 --- Determination of amino acid composition and N-terminal sequence of r-gcGH --- p.32 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Recombinant plasmids construction --- p.34 / Chapter 3.1.1 --- Basic construction of plasmid producing gcGH in E.coli --- p.34 / Chapter 3.1.2 --- N-terminal modification of gcGH cDNA --- p.38 / Chapter 3.1.3 --- Constuction of a short 'dummy' cistron at the 5'end of gcGH cDNA --- p.40 / Chapter 3.1.4 --- Optimization of distance between ribosomal binding site and initiation codon --- p.42 / Chapter 3.1.5 --- Increasing expression level by increasing plasmid copy number --- p.44 / Chapter 3.1.6 --- Optimizing the high density expression by changing the promoter --- p.48 / Chapter 3.1.7 --- Construction of excretion plasmid for gcGH production from E. coli --- p.48 / Chapter 3.2 --- Quantitation and qualitation of the expressed protein --- p.51 / Chapter 3.3 --- Effect of IPTG on induction of r-gcGH in pp5 --- p.57 / Chapter 3.4 --- Stability of overproducing strain pp5 during continuous culture --- p.59 / Chapter 3.5 --- Stability of overproducing strain ppADH4 during continuous culture --- p.61 / Chapter 3.6 --- "Optimization of culture condition for high level expression strains,pp5 and ppADH4" --- p.64 / Chapter 3.7 --- Purification of r-gcGH --- p.67 / Chapter 3.7.1 --- Distribution of r-gcGH as Soluble and insoluble protein in E. coli --- p.67 / Chapter 3.7.2 --- Isolation and cleaning of the inclusion bodies --- p.69 / Chapter 3.7.3 --- Solubilization and renaturation of r-gcGH --- p.71 / Chapter 3.7.4 --- Purification of r-gcGH by chromatography --- p.73 / Chapter 3.8 --- Characterization of r-gcGH --- p.78 / Chapter 3.8.1 --- Amino acid composition and N-terminal sequence determination --- p.78 / Chapter 3.8.2 --- Immunological property of r-gcGH --- p.81 / Chapter 3.8.3 --- Physical Property of r-gcGH --- p.84 / Chapter 3.8.4 --- Stability of r-gcGH --- p.84 / Chapter 3.9 --- Expression and purification of r-gcGH in excretion vector ppSP14 --- p.86 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Evaluation of expression strains --- p.88 / Chapter 4.1.1 --- Strain pKgcGH2 --- p.88 / Chapter 4.1.2 --- Strain pKgcGH2-17 --- p.88 / Chapter 4.1.3 --- Strain pSD78 --- p.89 / Chapter 4.1.4 --- "Strains pLl,pL2 and pL4" --- p.90 / Chapter 4.1.5 --- "Strains pp5,pplA,pp2I and pp4Q" --- p.90 / Chapter 4.1.6 --- Strain ppADH4 --- p.91 / Chapter 4.1.7 --- Strain ppSP14 --- p.91 / Chapter 4.2 --- Disulfide bond formation during refolding process --- p.92 / Chapter 4.2.1 --- Renaturaion in the presence of L-arginine and thiol reagent in oxidized form --- p.93 / Chapter 4.2.2 --- Renaturation in the presence of thiol reagent and 3M guanidine hydrochloride --- p.94 / Chapter 4.3 --- Stability of the r-gcGH --- p.94 / Chapter 4.4 --- Further studies --- p.96 / References --- p.98

Yu, Man Ying Susana. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-117). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of contents --- p.viii / Symbols and abbreviations --- p.xii / Scientific names --- p.xiv / List of figures --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Regulation of ovarian follicle development --- p.3 / Chapter 1.2.1 --- Endocrine regulation --- p.3 / Chapter 1.2.1.1 --- Gonadotropins- FSH and LH --- p.3 / Chapter 1.2.1.2 --- Co-gonadotropin- growth hormone --- p.5 / Chapter 1.2.2. --- Paracrine regulation --- p.6 / Chapter 1.2.2.1 --- Activin --- p.6 / Chapter 1.2.2.2 --- Insulin-like growth factor I (IGF-I) --- p.7 / Chapter 1.3 --- The GH-IGF-I axis --- p.7 / Chapter 1.3.1 --- The somatomedin hypothesis --- p.8 / Chapter 1.3.2 --- "Structure and signaling of GH, GHR" --- p.8 / Chapter 1.3.3 --- Structure and signaling of IGF system --- p.9 / Chapter 1.3.4 --- Role of GH-IGF system in reproduction --- p.11 / Chapter 1.3.5 --- GH action in ovarian functions --- p.12 / Chapter 1.3.6 --- IGF-I action in ovarian functions --- p.13 / Chapter 1.3.7 --- The mini GH-IGF axis within the ovary --- p.14 / Chapter 1.4 --- Objectives of present study --- p.14 / Chapter Chapter 2 --- "Expression Profiles of the GH-IGF System in the Ovary of Zebrafish, Danio rerio" --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Material and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of tissues and different stages of follicles from the zebrafish --- p.22 / Chapter 2.2.3 --- Separation of somatic follicle layers and oocytes --- p.22 / Chapter 2.2.4 --- Primary follicle cell culture --- p.22 / Chapter 2.2.5 --- Total RNA extraction --- p.23 / Chapter 2.2.6 --- Reverse transcription --- p.23 / Chapter 2.2.7 --- "Validation of semi-quantitative RT-PCR assays for GH (gh), GHR (ghr), IGF-I (igf1), IGF-II (igf2), and IGF-I receptor (igf1r)" --- p.24 / Chapter 2.2.8 --- Data analysis --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.25 / Chapter 2.3.2 --- Spatial expression of GH-IGF in different tissues of zebrafish --- p.26 / Chapter 2.3.3 --- "Localization of gh, ghr, igf1, igf2 and igf1r within the zebrafish follicle" --- p.26 / Chapter 2.3.4 --- Temporal expression profiles of GH-IGF system during folliculogenesis --- p.28 / Chapter 2.4 --- Discussion --- p.28 / Chapter Chapter 3 --- Regulation of the GH-IGF-I System and Its Cross-talk with the Activin System in the Zebrafish Ovary --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Material and methods --- p.45 / Chapter 3.2.1 --- Animals --- p.45 / Chapter 3.2.2 --- Chemicals and hormones --- p.45 / Chapter 3.2.3 --- Primary follicle cell culture --- p.45 / Chapter 3.2.4 --- Preparation of ovarian fragments --- p.45 / Chapter 3.2.5 --- Total RNA extraction --- p.45 / Chapter 3.2.6 --- RT-PCR --- p.47 / Chapter 3.2.7 --- Construction of real-time PCR standards --- p.47 / Chapter 3.2.8 --- Real-time PCR and semi-quantitative RT-PCR --- p.48 / Chapter 3.2.9 --- Data analysis --- p.49 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- "Expression of growth hormone (gh), growth hormone receptors (ghr1 and ghr2\ IGF-I (igf1), IGF-II (igf2), IGF-I receptor (igf1ra and igf1rb), activin subunits (inhba and inhbb) and follistatin (fst) in cultured zebrafish ovarian fragments" --- p.49 / Chapter 3.3.2 --- "Establishment of real-time RT-PCR for zebrafish inhba, inhbb and bactin" --- p.50 / Chapter 3.3.3 --- GH regulation of activin expression in cultured zebrafish follicle cells --- p.50 / Chapter 3.3.4 --- GH regulation of IGF-I in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.5 --- IGF-I regulation of activin expression in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.6 --- Activin regulation of IGF system --- p.52 / Chapter 3.4 --- Discussion --- p.52 / Chapter Chapter 4 --- Production of recombinant zebrafish growth hormone --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Material and Methods --- p.71 / Chapter 4.2.1 --- Animals --- p.71 / Chapter 4.2.2 --- Construction of expression plasmids pPIC9K/zfGH --- p.71 / Chapter 4.2.3 --- Production of recombinant zebrafish GH using Pichia pastoris --- p.73 / Chapter 4.2.4 --- SDS-PAGE and silver staining --- p.74 / Chapter 4.2.5 --- Purification --- p.74 / Chapter 4.2.6 --- Primary follicle cell culture --- p.75 / Chapter 4.2.7 --- Zebrafish hepatic cell culture --- p.76 / Chapter 4.2.8 --- RNA extraction and RT-PCR --- p.76 / Chapter 4.2.9 --- Real-time PCR --- p.77 / Chapter 4.2.10 --- Cell culture and transient transfection --- p.78 / Chapter 4.2.11 --- Luciferase reporter gene assay --- p.78 / Chapter 4.2.12 --- Data analysis --- p.79 / Chapter 4.3 --- Results --- p.79 / Chapter 4.3.1 --- Production of recombinant zebrafish GH --- p.79 / Chapter 4.3.2 --- Effect of recombiant zfGH on the expression of activin β Aand βB in cultured zebrafish follicle cells --- p.80 / Chapter 4.3.3 --- Effect of zfGH on the expression of igf1 in cultured zebrafish hepatic cells --- p.80 / Chapter 4.3.4 --- Luciferase reporter gene assay --- p.81 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- General Discussion --- p.94 / Chapter 5.1 --- Overview --- p.94 / Chapter 5.2 --- Major achievements of the present study --- p.95 / Chapter 5.2.1 --- Demonstration of a local mini-GH-IGF-I axis within the zebrafish ovary --- p.96 / Chapter 5.2.2 --- Differential expression profiles of the GH-IGF system during folliculogenesis --- p.96 / Chapter 5.2.3 --- The inter-relationship of GH-IGF and activin-follistatin systems --- p.96 / Chapter 5.2.4 --- Production of recombinant zebrafish GH --- p.97 / Chapter 5.3 --- Future prospects --- p.97 / References --- p.102 / Symbols and Abbreviations / Symbols / α Alpha / β Beta / Abbreviations / 20β-HSD 20β-hydroxysteroid dehydrogenase / bp Base pair / cAMP Cyclic adenosine monophosphate / cDNA Complementary cDNA / CHO Chinese hamster ovary / "DHP 17α, 20β-dihydroxy-4-prenane-3 -one" / DNA Deoxyribonucleic acid / EGF Epidermal growth factor